The present invention is directed to a method and device for forming sample wells in an electrophoresis gel slab. More particularly, the invention is directed to a method and device for forming a plurality of angled sample wells along a vertical edge of a gel slab for retaining liquid samples during a first dimension electrophoresis.
The separation of proteins and other macromolecules is often carried out by a slab gel electrophoresis separation in which the sample migrates through the gel slab, to a point determined by the molecular weight of the molecules in the sample. The gel acts as a sieve to separate the molecules according to their molecular weight.
This separation is often performed by a sodium dodecyl sulfate electrophoresis process within a two-dimensional acrylamide gel slab. A gel-forming composition is placed between two glass plates. The glass plates include spacers at opposite side edges to form a uniform gap between the plates. A seal may be placed along the open bottom edge, and the gap is filled with the gel-forming composition or the plates filled while in an enclosing box. The gel forming composition is allowed to set and the sample is placed along the edge of the gel slab. The protein sample may be a spaghetti-like strip of an isoelectric focusing gel that has been removed from a gel tube after a first dimension electrophoresis process. Alternatively, a liquid sample can be placed in a well that is formed in the end of the gel slab.
The open ends of the gel slab are placed in contact with buffer solutions and an electric potential is applied between the ends of the gel slab to cause the molecules to migrate through the gel slab. The gel slabs can be oriented vertically so that the molecules migrate downward through the gel slab.
A suitable apparatus for electrophoresis separation on a gel slab is disclosed in U.S. Pat. No. 4,088,561 to Anderson which is hereby incorporated by reference in its entirety. This apparatus contains a tank for supporting a plurality of slab gels supported between glass plates. Non-liquid samples are placed along the side edge of the gel slab so that the molecules migrate from one side edge to the opposite side edge in a horizontal direction. Seals are provided in the tank along the opposite side edges of the glass plates to contain and isolate the respective buffer solution at each end of the gel slab. Electrodes are provided at opposite ends to apply the electric potential between the open ends of the gel slab.
Slab gels are also used for separation of liquid samples that have been prepared by various techniques. Liquid samples are often prepared by centrifuging the protein sample to isolate certain cell fractions based on the separation of layers by the centrifuge or by solubilization of whole tissues or body fluids. Indentations or wells are formed along the edge of the gel slab to receive the liquid sample during the electrophoresis separation. Typically, the sample wells are formed in the end of the gel slab or in the end of an attached stacking gel by inserting a notched device into the end of the gel forming material and allowing the gel to set around the notches. The notches are typically square teeth that are inserted straight into the gel perpendicular to the edge of the gel slab. The device is then removed from the gel to provide the sample wells.
One example of a device, referred to as a comb, for forming wells in a gel slab is disclosed in U.S. Pat. No. 5,164,065 to Bettencourt et al. The comb includes a plurality of sharp teeth extending from a plate. The teeth are inserted into the bottom edge of a gel for forming lanes for separating the samples. The comb is removed from the gel and placed along the top edge of the gel. The spaces between the teeth receive the samples for separation.
The electrophoresis gels are fragile and can stick to the comb or well-forming device as the gel sets. It is often difficult to remove the gel-forming device without damaging or tearing portions of the gel slab. The suction effect caused by removing the teeth from the gel often causes the wells to collapse and distort. The result is a gel slab that has an inconsistent number of sample wells and sample wells that are different shapes and sizes due to the distortion and tearing of the gel. The wells are formed in the edge of the gel slab so that the gel slab cannot be rotated 90xc2x0 without spilling the liquid sample.
In view of the deficiencies of the above-noted devices, there is a continuing need in the industry for an improved device for forming sample wells in an electrophoresis gel slab.
The present invention is directed to a method and device for forming sample wells in the edge of an electrophoresis gel slab. More particularly, the invention is directed to a method and device for forming angled sample wells along a vertical edge of the gel slab for retaining liquid samples in a slab gel electrophoresis separation in which the gel is placed in an orientation such that the electrophoretic movement occurs in a horizontal direction.
Accordingly, a primary object of the invention is to provide a method and device for forming a plurality of spaced-apart sample wells along the vertical edge of a gel slab for containing a plurality of liquid samples.
Another object of the invention is to provide a method and device for forming sample wells along the edge of a gel slab substantially without distorting or tearing the gel material.
Still another object of the invention is to provide a method and device for forming sample wells in gel slabs for use in a DALT electrophoresis system.
A further object of the invention is to provide a sample well-forming device that can be removed from an electrophoresis gel-forming material after the gel has set without tearing the gel or collapsing the sample wells formed in the gel.
Another object of the invention is to provide a method and device for forming sample wells along the side edge of a gel slab for use in an automated second dimension electrophoresis process where the sample wells are formed to contain a liquid sample.
A further object of the invention is to provide a device for forming sample wells in a gel slab where the device has a plurality of projections that have a rounded tip such that the projections can be removed from the gel material after the gel has polymerized without tearing or distorting the sample wells formed in the gel by the projections.
Still another object of the invention is to provide a device for forming angled sample wells in a gel slab, where the device has a plurality of angled teeth having sides that converge to a rounded tip.
Another object of the invention is to provide a device for forming sample wells in a gel slab where the device has a plurality of spaced-apart projections having an axial passage for allowing air into the sample well formed in a gel slab as the projections are removed from the gel slab, thereby preventing collapse of the sample well formed in the gel.
A further object of the invention is to provide a device for forming a plurality of sample wells along the edge of a gel slab where the device has a plurality of projections having an axial passage and a removable pin received in the axial passage of each of the projections that can be removed after the gel has polymerized and before the projections are removed from the gel.
The objects of the invention are basically attained by providing a device for forming a plurality of sample wells in an electrophoresis gel slab. The device comprises a body having a longitudinal edge with a longitudinal dimension, and a plurality of uniformly spaced-apart projections extending from the longitudinal edge of the body at an incline with respect to the longitudinal dimension. The projections are oriented in a row and extend substantially parallel to each other. The projections have a dimension to form a sample well in a side edge of an electrophoresis gel slab.
The objects of the invention are further attained by providing a device for forming angled sample wells along a vertical edge of a gel slab. The device comprises a body having a longitudinal dimension and a longitudinal side edge, and a plurality of projections integrally formed with the body and having a dimension for forming sample wells in a side edge of an electrophoresis gel slab. The projections extend from the longitudinal side edge at an angle of about 30xc2x0 to about 50xc2x0 with respect to the longitudinal dimension. The projections further have a tip and first and second side edges converging to the tip. The first and second side edges are oriented at an angle with respect to each other to enable the projections to be removed from an electrophoresis gel substantially without distortion of the gel.
The objects of the invention are further attained by providing a method of forming an electrophoresis gel slab having a plurality of liquid sample wells. The process comprises the steps of: providing a gel-forming substance between two spaced apart supporting plates having an open side edge and positioning a well-forming device for forming sample wells in the open side edge of the supporting plates and into the gel-forming substance. The device has a body with a longitudinal edge with a longitudinal dimension, and a plurality of uniformly spaced apart projections extending from the body at an incline with respect to the longitudinal dimension. The gel forming substance is solidified to form an electrophoresis gel slab which has a side edge. The well-forming device is removed from the glass plates to form an electrophoresis gel slab between the supporting plates. The gel slab has a plurality of sample wells along the edge of the gel slab. Each of the sample wells is formed at an angle with respect to a longitudinal dimension of the side edge of the gel slab for retaining a liquid sample when the side edge of the gel slab is oriented vertically.
The objects and advantages are also attained by providing a process for separating substances by second dimension electrophoresis. The process comprises the steps of: forming an electrophoresis gel slab having a first longitudinal edge and a plurality of spaced-apart sample wells formed in the gel. The wells have an axial dimension defining a depth. The axial dimension of the wells is oriented at an incline with respect to the longitudinal edge of the gel slab. The gel slab further has a second longitudinal edge. Test liquid samples are placed in each of the sample wells. The gel slab is positioned in a second dimension electrophoresis tank with the longitudinal edge of the gel slab oriented vertically and the sample wells are positioned in the gel slab at an incline with respect to the longitudinal edge to retain the liquid samples in the wells. First and second longitudinal edges of the gel slab are contacted with a buffer solution which is less dense than the liquid samples permitting them to remain in the bottoms of the wells in hydrostatic equilibrium and an electric potential is applied between the first and second longitudinal ends of the gel slab to cause molecules in the samples to migrate through the gel slab.
The objects, advantages and salient features of the invention will become apparent to one skilled in the art in view of the following detailed description of the invention in conjunction with the annexed drawings which form a part of this original disclosure.